CN113720820A - β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用 - Google Patents
β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用 Download PDFInfo
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Abstract
本发明公开了一种β‑葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用，该传感器中蛋白‑无机杂交纳米花可通过电子转移将荧光红染料催化转化为深紫红色，具有强荧光的试卤灵。当向体系中加入β‑Glu时，由于生氰苷的存在，β‑Glu能够特异性水解生氰苷，并生成CN‑。CN‑能够有效地抑制蛋白‑无机杂交纳米花的催化活性，使得体系的荧光强度减弱，同时伴随着溶液颜色变浅。通过监测体系的颜色变化以及荧光的“Turn on‑off”，可以实现β‑Glu的光学双信号检测。所构建荧光纳米传感器还可用于土壤中β‑Glu的定量检测。此外，该传感器在筛选β‑Glu抑制剂方面具有一定的应用潜力。
Description
技术领域
本发明公开涉及β-葡萄糖苷酶活性检测的技术领域，尤其涉及一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用。
背景技术
β-葡萄糖苷酶(β-Glu)，一类能够特异性催化β-糖苷键水解的糖基水解酶，对植物、动物和微生物产生功能性次生代谢产物糖苷具有重要意义。近二十年来，β-Glu已被广泛应用于医药、生物能源、环境、食品等领域。例如，β-Glu活性水平与糖尿病、细菌或病毒感染和癌症等代谢性疾病有关[Lillelund,V.H.；Jensen,H.H.；Liang,X.；Bols,M.Recentdevelopments of transition-state analogue glycosidase inhibitors of non-natural product origin.Chem Rev,2002,102,515-553]。在生物技术方面，β-Glu活性在从可再生木质纤维素生产第二代和第三代环境友好型生物燃料和化学品中起着极其重要的作用[Huang,D.L.；Zeng,G.M.；Feng,C.L.；Hu,S.；Jiang,X.Y.；Tang,L.；Su,F.F.；Zhang,Y.；Zeng,W.；Liu,H.L.Degradation of lead-contaminated lignocellulosic waste byphanerochaete chrysosporium and the reduction of lead toxicity.Environ SciTechnol,2008,42,4946-4951]。在环境方面，已有研究表明富营养化湖泊中β-Glu活性与春季浮游植物水华大量繁殖密切相关[Chróst,R.J.；Overbeck,J.Substrate-ectoenzymeinteraction:significance of β-glucosidase activity for glucose metabolism byaquatic bacteria.Arch Hydrobiol Beih Ergebn Limnol,1990,34,93-98]。在农业方面，近年来也有令人兴奋的发现，β-Glu活性可以作为土壤质量的生物学指标[Sazawa,K.；Kuramitz,H.Hydrodynamic voltammetry as a rapid and simple method forevaluating soil enzyme activities.Sensors,2015,15,5331-5343]。因此，检测β-Glu和监测β-Glu活性具有重要意义。
目前，传统测定β-Glu活性的方法是基于底物(对硝基苯基-β-D-吡喃葡萄糖苷)转化为对硝基苯酚，继而用分光光度法定量。然而，该方法的一个重要缺陷是用于检测β-Glu活性的底物非常不稳定；另外，该方法涉及大量对环境非常有害的试剂[Stege,P.W.；Messina,G.A.；Bianchi,G.；Olsina,R.A.；Raba,J.Determination of β-glucosidaseactivity in soils with a bioanalytical sensor modified with multiwalledcarbon nanotubes.Anal Bioanal Chem,2010,397,1347-1353]。最近，报道的一些新型荧光检测β-Glu活性的方法大多基于苯酚衍生物的β-糖苷，如硝基酚[Yan,S.；Wu,G.Prediction of michaelis-menten constant of beta-glucosidases usingnitrophenyl-beta-D-glucopyranoside as substrate.Protein Pept Lett,2011,18,1053-1057]、荧光素[Stege,P.W.；Messina,G.A.；Bianchi,G.；Olsina,R.A.Determinationof the β-glucosidase activity in different soils by pre capillary enzymeassay using capillary electrophoresis with laser-induced fluorescencedetection.J Fluoresc,2010,20,517-523]和7-羟基香豆素[Watanabe,A.；Suzuki,M.；Ujiie,S.；Gomi,K.Purification and enzymatic characterization of a novel β-1,6-glucosidase from Aspergillus oryzae.J Biosci Bioeng,2016,121,259-264]。但此类方法中使用的荧光团的蓝色荧光与生物样品背景重叠，在测定酶活性之前需要对其进行分离纯化，导致β-Glu活性的分析复杂化。此外，检测耗时或重复性差等问题也使得这些检测方法的应用受到进一步限制。
因此，迫切需要设计一种操作简单、高灵敏度、有效、低成本且环保的β-Glu活性检测试剂。
发明内容
鉴于此，本发明提供了一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用，以解决以往在β-葡萄糖苷酶活性检测时，存在操作复杂，有效性差等问题。
一方面，本发明提供了一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，该免标记型光学纳米传感器由以下成分组成：具有仿酶活性的蛋白-无机杂交纳米花、荧光红染料、生氰苷以及过氧化氢溶液。
优选，所述具有仿酶活性的蛋白-无机杂交纳米花为BSA-Cu3(PO4)2·3H2O NFs。
进一步优选，所述荧光红染料的浓度为20μmol·L-1。
进一步优选，所述生氰苷为苦杏仁苷溶液。
进一步优选，所述苦杏仁苷溶液的浓度为20mmol·L-1。
进一步优选，过氧化氢溶液的浓度为200mmol·L-1。
另一方面，本发明还提供了一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器的应用，具体为，用于土壤中β-Glu的检测以及β-Glu抑制剂的筛选。
本发明提供的β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，传感器中的蛋白-无机杂交纳米花可以通过电子转移将荧光红染料催化转化为深紫红色，具有强荧光的试卤灵(Resorufin)。当向体系中加入β-Glu时，由于生氰苷的存在，β-Glu能够特异性水解生氰苷，并生成氰根离子(CN-)。CN-能够有效地抑制蛋白-无机杂交纳米花的催化活性，使得体系荧光强度的减弱，同时伴随着溶液颜色变浅。CN-的浓度由不同浓度的β-Glu触发的酶解反应控制，因此通过监测体系的颜色变化以及荧光的“Turn on-off”，可以实现β-Glu的光学双信号检测。在最佳条件下，所构建光学纳米传感器在0.5-1500U·L-1对β-Glu的检测表现出良好线性关系；检测限为0.33U·L-1。所构建荧光纳米传感器还可用于土壤中β-Glu的定量检测。此外，该传感器在筛选β-Glu抑制剂方面具有一定的应用潜力。
与现有技术相比，本发明所建立的光学纳米传感器是基于具有仿酶活性的蛋白-无机杂化纳米花，使得所构筑的传感器具有制备简单，成本低廉，催化活性强，且不易受环境条件影响等诸多优势。另外，本发明所述的光学纳米传感器无需标记，只是利用电子转移诱导荧光猝灭和特殊酶解诱导荧光恢复来触发光学响应，避免了多步功能化或耗时、劳动密集型的复杂标记过程。再者，本发明所述的光学传感器实现了双信号检测，使其具备更有潜力应用前景。所述荧光纳米传感器对β-Glu活性呈现出较宽的线性响应范围，检出限也较低。
应当理解的是，以上的一般描述和后文的细节描述仅是示例性和解释性的，并不能限制本发明的公开。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本发明的实施例，并与说明书一起用于解释本发明的原理。
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明所提出的(A)自组装合成BSA-Cu3(PO4)2·3H2O NFs，(B)H2O2存在条件下BSA-Cu3(PO4)2·3H2O NFs催化氧化Amplex Red为resorufin，(C)免标记双信号光学纳米传感器检测β-Glu活性的示意图；
图2分别为AR空白对照(a),BSA-Cu3(PO4)2·3H2O NFs+AR混合溶液(b),H2O2+AR混合溶液(c),AR+BSA-Cu3(PO4)2·3H2O NFs+H2O2混合溶液(d),and AR+BSA-Cu3(PO4)2·3H2ONFs+H2O2+β-Glu+Amy混合溶液(e)各自的荧光发射光谱。插图为(a)、(b)、(c)、(d)和(e)溶液相应的颜色。FL是荧光的缩写，AR是Amplex Red的缩写；
图3为本发明BSA-Cu3(PO4)2·3H2O NFs与Amplex Red混合溶液在不同浓度H2O2(0-200.0mmol·L-1)存在条件下的荧光发射光谱图(A)；不同浓度H2O2与混合溶液荧光强度之间的点线关系图，插图为在5-60mmol·L-1范围内H2O2浓度与体系荧光强度之间的线性关系(B)；
图4为孵育时间对所制备BSA-Cu3(PO4)2·3H2O NFs催化性能的影响；
图5为pH对BSA-Cu3(PO4)2·3H2O NFs和HRP催化活性的影响；
图6为本发明所述的基于BSA-Cu3(PO4)2·3H2O NFs-H2O2-Amplex Red-Amy纳米传感器响应不同浓度β-Glu(0-1500.0U·L-1)的荧光发射光谱图(a)；β-Glu浓度与体系荧光强度比值(I/I0)之间的线性关系图，I与I0代表有无β-Glu存在条件下BSA-Cu3(PO4)2·3H2ONFs-H2O2-Amplex Red-Amy混合溶液的荧光发射光谱(b)；日光下不同β-Glu活性催化传感体系相应的颜色变化图(c)；
图7为本发明所述的基于BSA-Cu3(PO4)2·3H2O NFs-H2O2-Amplex Red-Amy光学纳米传感器对β-Glu检测的抗干扰能力与选择性研究。
具体实施方式
下面将结合具体实施例和附图对本发明作进一步详细说明，但是并不用于限制本发明的保护范围。
以下实施例中所有的化学药品和试剂至少为分析纯级别，所有实验使用的超纯水(18.2MΩ·cm-1)均来自Milli-Q Integral 10水纯化装置。
实施例1：BSA-Cu3(PO4)2·3H2O NFs的制备
BSA-Cu3(PO4)2·3H2O NFs具体合成路线如下[见图1(A)]：首先，将200mmol·L-1CuSO4溶液注入1690μL含有0.1mg·mL-1 BSA的50mmol·L-1、pH 6.8的磷酸盐缓冲溶液(PBS)中，然后在室温下静置12小时。将制备的BSA-Cu3(PO4)2·3H2O NFs在10000rpm下离心10min，然后用超纯水洗涤3次进行纯化。最后，将收集的蓝绿色沉淀重新分散于200μL PBS中，于4℃保存，以用于接下来的实验。
实施例2：BSA-Cu3(PO4)2·3H2O NFs催化性能研究
利用H2O2和Amplex Red溶液(荧光和显色底物)考察了BSA-Cu3(PO4)2·3H2O NFs的仿过氧化物酶催化性能。如图2所示，在对照实验中，Amplex Red溶液(曲线a)、Amplex Red与NFs混合溶液(曲线b)以及Amplex Red与H2O2混合溶液(曲线c)在565～800nm之间没有明显的荧光发射峰。当将BSA-Cu3(PO4)2·3H2O NFs、Amplex Red与H2O2三者混合时，混合溶液在584nm处表现出显著的荧光增强(曲线d)，该荧光来自于Amplex Red的氧化产物Resorufin，并且背景荧光极低，可以忽略不计。上述结果表明，BSA-Cu3(PO4)2·3H2O NFs具有优异的仿过氧化物酶催化活性。当激发波长为550nm时，反应体系的最大荧光发射波长约为584nm。同时，如图2的插图所示，可以观察到溶液由无色迅速转变为典型的深紫红色，肉眼很容易辨认。
为了进一步研究BSA-Cu3(PO4)2·3H2O NFs的催化性能，我们测试了一定量BSA-Cu3(PO4)2·3H2O NFs、Amplex Red与不同浓度H2O2溶液共存时，体系的荧光发射光谱。如图3所示，在0.05-200mmol·L-1浓度范围内，随着H2O2浓度的增加，体系荧光发射强度逐渐增强；从图3插图可以看出，体系荧光发射强度与H2O2浓度呈现出良好的线性关系。上述结果进一步证实了BSA-Cu3(PO4)2·3H2O NFs具有良好的类过氧化物酶催化性能。
众所周知，酶的催化性能与反应条件密切相关，尤其pH与孵育时间，因此本实施例考察了溶液pH与孵育时间对制备的BSA-Cu3(PO4)2·3H2O NFs酶催化性能的影响。从图4中可以看出，以Amplex Red作为底物，在pH范围为4.0-9.0时，酸性介质中BSA-Cu3(PO4)2·3H2O NFs催化活性和辣根过氧化物酶(HRP)催化活性均高于碱性介质，但BSA-Cu3(PO4)2·3H2O NFs总体上比HRP具有更好的催化性能。从图5可以看出，在一定范围内，体系荧光发射强度随着孵育时间的增加而增强，在90min左右达到平台，表明催化反应在90min内完成。
实施例3：光学纳米传感器的构建及其对β-Glu的响应
首先，制备20000U·L-1的β-Glu储备液。分别将150μL的NFs溶液和50μL的苦杏仁苷溶液(20mmol·L-1)加入一系列50μL不同浓度的β-Glu溶液，并充分混匀，室温孵育1h进行酶促反应。然后在上述反应溶液中分别加入20μmol·L-1 Amplex Red溶液和200mmol·L-1的H2O2溶液，用磷酸盐缓冲溶液(10mmol·L-1,pH＝6.8)定容至1.5mL。充分混匀后，在室温条件下孵育90分钟。于550nm激发光条件下，测定上述混合溶液在565-800nm范围内的荧光发射光谱，结果如图6所示。激发和发射的狭缝均为5nm。从图6中可以观察到，在0-1500.0U·L-1范围内，随着β-Glu含量的增加，BSA-Cu3(PO4)2·3H2O NFs-H2O2-Amplex Red-Amy体系在584nm处的荧光强度逐步降低，并且伴随着混合溶液的颜色由深紫红逐渐变浅至浅品红色[图6(c)]。另外，由图2可以估算出，BSA-Cu3(PO4)2·3H2O NFs-Amplex Red-H2O2体系在584nm处的荧光发射强度下降至原始强度的33.8％。同时，图6(b)显示β-Glu浓度在0.5-1500U·L-1范围内与荧光强度比率(I/I0，I与I0代表有无β-Glu存在条件下BSA-Cu3(PO4)2·3H2O NFs-H2O2-Amplex Red-Amy混合溶液的荧光发射光谱)存在良好的线性关系，线性回归方程为I/I0＝0.992-0.0002[β-Glu](U·L-1)(R2＝0.997)。β-Glu的检测限(LOD)为0.33U·L-1。与现有的β-Glu检测方法相比，本方法具有较低的LOD和较宽的定量范围。
实施例4：光学纳米传感器的选择性考察
选择性是评价一种新型传感器性能的重要参数，特别是对于在复杂环境样品中具有潜在应用价值的传感器，对目标的高度选择性响应非常有必要。因此，在添加Na+、K+、Ca2 +、Mg2+、NO3-和Cl-1、葡萄糖氧化酶(Glu)、葡萄糖氧化酶(GOX)、酪氨酸酶(TYR)和酸性磷酸酶(ACP)、L-半胱氨酸、葡萄糖和鱼精蛋白等多种常见共存物质的情况下，对所述的光学纳米传感器进行了选择性实验，结果如图7所示。ACP的浓度为1.0μU·mL-1。GOX的浓度为1.0μg·mL-1。L-半胱氨酸、葡萄糖和鱼精蛋白的浓度为1.0μmol·L-1。Na+、K+、Ca2+、Mg2+、NO3-和Cl-1的浓度为1.0μmol·L-1。从图7可以看出，所发明的光学纳米传感器不仅对其他可能共存的物质无响应，而且对其他可能共存的物质存在时的β-Glu依然具有选择性，说明其抗干扰能力强。
实施例5：土壤溶液中β-Glu活性的检测
本实施例所使用土壤由当地生物地球化学实验室提供，所有土壤均为表层土(0～20cm)。清除土壤样品中植物的根和其他杂质后，过2mm筛。取1.0g土壤样品溶于15mL超纯水中，室温震荡1小时；然后，于12000rpm离心10分钟，过滤得到上清液。将不同浓度的β-Glu添加到土壤样品中，制备添加样品，接着按照实施例3中所述步骤检测土壤样品中β-Glu活性，结果如表1所示。采用标准加样法进行回收率试验，根据标准曲线和回归方程推导出样品中β-Glu的活性，RSD一般从3个平行样品中计算获得。从表1可以看出，β-Glu的平均加标回收率为96.2-104％，RSD小于5.0％。因此，所述的传感系统的精度和精度是令人满意的。
表1：光学纳米传感器在实际土壤样品中的检测结果
本领域技术人员在考虑说明书及实践这里公开的发明后，将容易想到本发明的其它实施方案。本申请旨在涵盖本发明的任何变型、用途或者适应性变化，这些变型、用途或者适应性变化遵循本发明的一般性原理并包括本发明未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的，本发明的真正范围和精神由下面的权利要求指出。
应当理解的是，本发明并不局限于上面已经描述的内容，并且可以在不脱离其范围进行各种修改和改变。本发明的范围仅由所附的权利要求来限制。
Claims (7)
1.一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，由以下成分组成：具有仿酶活性的蛋白-无机杂交纳米花、荧光红染料、生氰苷以及过氧化氢溶液。
2.根据权利要求1所述β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，所述具有仿酶活性的蛋白-无机杂交纳米花为BSA-Cu3(PO4)2·3H2O NFs。
3.根据权利要求1或2所述β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，所述荧光红染料的浓度为20μmol·L-1。
4.根据权利要求1或2所述β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，所述生氰苷为苦杏仁苷溶液。
5.根据权利要求4所述β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，所述苦杏仁苷溶液的浓度为20mmol·L-1。
6.根据权利要求1或2所述β-葡萄糖苷酶活性检测用免标记型光学纳米传感器，其特征在于，过氧化氢溶液的浓度为200mmol·L-1。
7.一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器的应用，其特征在于，用于土壤中β-Glu的检测以及β-Glu抑制剂的筛选。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5792662A (en) * | 1994-11-08 | 1998-08-11 | Tosoh Corporation | Method of determination of fluorescent substance and method of assay of enzyme activity |
| US20150126409A1 (en) * | 2013-11-01 | 2015-05-07 | Chemgreen Innovation Inc. | Nanostructured microbial sensors |
| CN107602643A (zh) * | 2017-09-25 | 2018-01-19 | 王铮 | 一种基于萘酰亚胺的β‑葡萄糖醛酸苷酶的荧光探针及其应用 |
| CN109557065A (zh) * | 2019-01-08 | 2019-04-02 | 吉林省农业科学院 | 一种检测土壤中β-D-葡萄糖苷酶活性的分析方法 |
| CN111504987A (zh) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | 一种利用无机杂化纳米花酶快速检测二胺类生物胺的方法 |
| KR20210081915A (ko) * | 2019-12-24 | 2021-07-02 | 중앙대학교 산학협력단 | 포도당 검출을 위한 바이오센서의 전극 및 이의 제조방법 |
-
2021
- 2021-09-03 CN CN202111030203.1A patent/CN113720820A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5792662A (en) * | 1994-11-08 | 1998-08-11 | Tosoh Corporation | Method of determination of fluorescent substance and method of assay of enzyme activity |
| US20150126409A1 (en) * | 2013-11-01 | 2015-05-07 | Chemgreen Innovation Inc. | Nanostructured microbial sensors |
| CN107602643A (zh) * | 2017-09-25 | 2018-01-19 | 王铮 | 一种基于萘酰亚胺的β‑葡萄糖醛酸苷酶的荧光探针及其应用 |
| CN109557065A (zh) * | 2019-01-08 | 2019-04-02 | 吉林省农业科学院 | 一种检测土壤中β-D-葡萄糖苷酶活性的分析方法 |
| KR20210081915A (ko) * | 2019-12-24 | 2021-07-02 | 중앙대학교 산학협력단 | 포도당 검출을 위한 바이오센서의 전극 및 이의 제조방법 |
| CN111504987A (zh) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | 一种利用无机杂化纳米花酶快速检测二胺类生物胺的方法 |
Non-Patent Citations (2)
| Title |
|---|
| ZIPING LIU 等: "A Label-free Optical Sensing Platform for Beta-glucosidase Activity Using Protein-inorganic Hybrid Nanoflowers", RESEARCH SQUARE, pages 1 - 19 * |
| 廖小晴 等: "过氧化氢与L-半胱氨酸对BSA-Cu体系荧光"开-关"响应及分析应用", 分析化学, vol. 43, no. 12, pages 1820 - 1828 * |
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